In our continued efforts to study the host response to influenza A virus infection, we performed deep sequencing on small RNAs from infected cells and discovered a large population of influenza A virus derived small RNAs mapping to the 5'end of the each of the viral RNA segments. Expression of this small RNA was corroborated by northern blot and required the viral polymerase. Given the known secondary structure of the viral genome, we hypothesized that generation of this small viral RNA (svRNA) would result in disruption of the panhandle and would cause a switch from transcription to replication. As the timing of svRNA production supported this hypothesis, we synthesized svRNA and, upon transfection, observed a decrease in mRNA production, an increase in vRNA synthesis, and a general loss of influenza virus propagation. Furthermore, blocking svRNA availability through competitive hybridization resulted in an increase in mRNA synthesis and complete loss of vRNA. Identification of svRNA answers a very longstanding question as to how influenza A virus switches from transcription to replication and exemplifies a new paradigm for small RNAs and virus infection. Despite determining this putative function for svRNA, how it is generated and the breadth of its activity remains largely unknown. Here we seek to understand these details more thoroughly. Aim 1 details a strategy to ascertain how each segment-specific svRNA functions. Aim 2 describes efforts to develop a model for in vitro svRNA synthesis to determine the molecular mechanism by which they are synthesized. Aim 3 investigates how svRNA affects the host cell environment. The experimental strategy comprising these aims will reveal exciting new molecular targets that can be exploited to generate a novel class of anti-influenza virus therapeutics and will significantly increase our understanding of influenza virus replication. PUBLIC HEALTH RELEVANCE: Greater understanding of influenza virus replication is imperative to control the virus's propensity to cause yearly epidemics and sporadic pandemics. This proposal focuses on the study of a novel small RNA component of the virus in an effort to reveal novel molecular targets for anti-viral design.